cpf1-based genome editing using ribonucleoprotein complexes
TRANSCRIPT
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Cpf1-based genome editing using the Alt-R™ CRISPR-Cpf1 System
Rolf Turk, PhD
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Outline: Alt-R™ CRISPR-Cpf1 System
• Background• Optimization of Cpf1 crRNA
• Length optimization• Chemical modification
• Delivery of Cpf1 as ribonucleoprotein (RNP) complex• Effect of Alt-R Cpf1 Electroporation Enhancer• RNP concentration optimization
• Homology-directed repair using Cpf1• Positive controls
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Cas9 genome editing
• RNA-guided endonuclease• PAM site (NGG)• crRNA and tracrRNA• Blunt-ended cut sites
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Cpf1 genome editing
• RNA-guided endonuclease• Cpf1: CRISPR from Prevotella and Francisella 1• Class II, type V• Cpf1 editing in mammalian cells
• Acidaminococcus sp. BV3L6• Lachnospiraceae bacterium ND2006
• Single guide RNA (crRNA, 41–44 nt)• Double-stranded break with staggered ends• PAM site is thymidine-rich
• Preferentially uses TTTV
Zetsche B, Gootenberg JS, et al. (2015) Cpf1 is a single RNA-guided Endonuclease of a class 2 CRISPR-Cas system. Cell, 163:759–771.
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Cpf1 structure and function
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Yamano T, Nishimasu H, et al. (2016) Crystal structure of cpf1 in complex with guide RNA and target DNA. Cell, 165(4):949–962.
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Comparison of Cas9 and Cpf1
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Low off-target editing with Cpf1
Kim D, Kim J, et al. (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotech, 34(8):863–868.
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Lower off-target effects with Cpf1 RNP vs. plasmid
Kim D, Kim J, et al. (2016) Genome-wide analysis reveals specificities of cpf1 endonucleases in human cells. Nat Biotech, 34(8):863–868.
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Alt-R™ A.s. Cpf1 Nuclease 2NLS on-target efficiency
46550 46650 46750 46850 46950 47050 47150 472500
102030405060708090
100
STAT3: exons 5 and 6HEK 293—RNP—Amaxa® Nucleofector® System
AsCpf1 SpCas9
Chromosome location
T7EI
tota
l edi
ting
effici
ency
(%)
0 10 20 30 40 50 60 70 80 90 1000
102030405060708090
100
STAT3: exons 5 and 6HEK 293—RNP—Amaxa® Nucleofector® System
AsCpf1 SpCas9
Ranked editing efficiency
T7EI
tota
l edi
ting
effici
ency
(%)
93%
35%15%
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A.s. Cpf1 editing efficiency is PAM-sequence dependent
0 10 20 30 40 50 60 70 800102030405060708090
100
HEK 293—RNP—Amaxa® Nucleofector® System232 crRNAs across 6 genes
TTTATTTCTTTGTTTT
T7E
I tot
al e
ditin
g ef
ficie
ncy
(%)
Ranked editing efficiency
Alt-R™ A.s. Cpf1 Nuclease 2NLS on-target efficiency
0 20 40 60 80 1000
10
20
30
40
50
60
70
80
90
100
STAT3: exons 5 and 6HEK 293—RNP—Amaxa® Nucleofector® System
AsCpf1 (TTTN) AsCpf1 (TTTV) SpCas9 (NGG)
Ranked editing efficiency
T7EI
tota
l edi
ting
efficie
ncy
(%)
93%
35%
15%
53%
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Alt-R™ CRISPR-Cpf1 RNP complex
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Alt-R™ CRISPR-Cpf1 crRNA—protospacer length optimization
38171-AS 38254-AS 38325-S 38337-AS 38351-S 38538-S0
102030405060708090
100
HEK 293–Cpf1 Stable Cell Line—30 nM crRNARNAiMAX™ (Thermo Fisher)
24 mer23 mer22 mer21 mer20 mer
HPRT1 crRNA location and guide strand
T7EI
tota
l edi
ting
effici
ency
(%)
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Alt-R™ CRISPR-Cpf1 crRNA—2′O-methyl testing
Locations affected by 2′OMe modification
T7EI total editing (%) T7EI total editing (%)
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Alt-R™ CRISPR-Cpf1 RNP complex formation
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Effect of Alt-R™ Cpf1 Electroporation Enhancer with RNP
38094-S
38104-S
38115-AS
38146-AS
38164-AS
38164-S
38186-S
38228-S
38330-AS
38343-S
38455-S
38486-S0
102030405060708090
100
HEK 293—5 µM RNP—Amaxa® Nucleofector® System
0 µM Enhancer3 µM Enhancer5 µM Enhancer
HPRT1 crRNA location and guide strand
T7EI
tota
l edi
ting
effici
ency
(%)
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Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Nucleofector®
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*
* = Toxicity
0 1 2 3 4 5 60
10
20
30
40
50
60
70
80
90
100
HEK 293—RNP—Amaxa® Nucleofector® System HPRT1 38228-S
No EnhancerEquimolar Enhancer3 µM Enhancer
Cpf1 ribonucleoprotein complex concentration (µM)
T7E
I tot
al e
ditin
g ef
ficie
ncy
(%)
20
0 1 2 3 4 5 60
10
20
30
40
50
60
70
80
90
100
HEK 293—RNP—Amaxa® Nucleofector® SystemHPRT1 38330-AS
No EnhancerEquimolar Enhancer3 µM Enhancer
Cpf1 ribonucleoprotein complex concentration (µM)
T7E
I tot
al e
ditin
g ef
ficie
ncy
(%)
** = Toxicity
Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Nucleofector®
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0 1 2 3 4 5 60
10
20
30
40
50
60
70
80
90
100
HEK 293—RNP—Neon® Transfection SystemHPRT1 38330-AS
No EnhancerEquimolar Enhancer1.8 µM Enhancer
Cpf1 ribonucleoprotein complex concentration (µM)
T7EI
tota
l edi
ting
effici
ency
(%)
Optimal Alt-R™ CRISPR-Cpf1 RNP concentration using Neon®
**
* = Toxicity
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Alt-R™ CRISPR-Cpf1 RNP editing efficiency—Nucleofector® vs. Neon® electroporation
38115-AS 38186-S 38228-S 38330-AS0102030405060708090
100
HEK 293 Cells
NFXNNeon
HPRT1 crRNA location and guide strand
T7EI
tota
l edi
ting
efficie
ncy
(%)
5 µM RNP3 µM Enhancer
5 µM RNP1.8 µM Enhancer
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Alt-R™ Cpf1 Electroporation Enhancer does not alter indel profile
Indel Size Distribution - NGS - HEK293
Indel size (bp)-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10
Sequ
ence
s (%)
0
5
60
80
1000 µM Cpf1 Electroporation Enhancer3 µM Cpf1 Electroporation Enhancer
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Alt-R™ CRISPR-Cpf1 editing—time course
15 25 35 45 55 65 750102030405060708090
100
Time post-transfection (hr)
T7E
I tot
al e
ditin
g ef
ficie
ncy
(%)
15 25 35 45 55 65 750102030405060708090
100
38115-AS38186-S38228-S38330-AS
Time post-transfection (hr)
HEK293 HeLa5 µM RNP—3 µM Enhancer—Amaxa® Nucleofector ® System
T7E
I tot
al e
ditin
g ef
ficie
ncy
(%)
25
Flanking arm
length
927257473727
Cpf1 cleavage
crRNA guide
GAATTC(EcoRI)
Total lengthssODN
190
150
120
100
80
60
Homology-directed repair in HEK 293–Cpf1 stable cell line
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Homology-directed repair in HEK 293–Cpf1 stable cell line
190
150
120
100 80 60 190
150
120
100 80 60
Non-targeted strand (nt) Targeted strand (nt)
0
20
40
60
80
100
HEK 293—HPRT1 38343-SRNAiMAX®—30 nM crRNA—30 ng HDR template (ssODN)
EcoRIT7
T7EI
tota
l edi
ting
effici
ency
(%)
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Alt-R™ CRISPR-Cpf1 RNP delivery using lipofection
1:1 2:1 5:1 1:1 2:1 5:1 1:1 2:1 5:110 nM Cpf1 30 nM Cpf1 50 nM Cpf1
0102030405060708090
100
HEK 293—RNAiMAX™
Cpf1 concentration and ratio crRNA:Cpf1
T7EI
tota
l edi
ting
effici
ency
(%)
* = Toxicity
* * * *
*
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Positive controls for genome editing using the Alt-R™ CRISPR-Cpf1 System
Hs - HEK 293 Mm - Hepa1-6 Rn - RAT20102030405060708090
100
5 µM RNP—3 µM EnhancerAmaxa® Nucleofector ® System
T7EI
tota
l edi
ting
effici
ency
(%)
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Comparison of genome editing using SpCas9 vs. AsCpf1
* Molecular weight of Alt-R™ Nuclease
† N = any base V = A, C, or GTTTV
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Alt-R™ CRISPR-Cpf1 System
Core components:• Alt-R CRISPR-Cpf1 crRNA• Alt-R A.s. Cpf1 Nuclease 2 NLS• Alt-R Cpf1 Electroporation EnhancerSequences for positive and negative crRNA controls for human, mouse, and rat are available at www.idtdna.com/CRISPR-Cpf1.
Also see our highly effective Alt-R CRISPR-Cas9 System at www.idtdna.com/CRISPR-Cas9.
THANK YOU
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